Salt-activated beta-galactosidase mutant d140g and use thereof

Abstract

The application discloses a salt-activated beta-galactosidase mutant D140G and application thereof, and belongs to the technical field of genetic engineering. In the application, the wild beta-galactosidase 6Y2K is mutated from aspartic acid at the 140th position to glycine, and a mutant D140G is obtained, and the amino acid sequence of the mutant D140G is shown as SEQ ID NO. 1. The mutant has very low activity without salt, is significantly activated in a salt solution of NaCl or KCl or the like with a concentration of 0.05M-3M, and the enzyme activity is increased by 103 times compared with that without salt; the salt activation effect is also presented on natural substrates such as lactose, and the enzyme reaction rate can be regulated by the salt concentration. The mutant provided by the application can realize dynamic regulation of the enzyme reaction rate by regulating the salt solution concentration, and is suitable for biological catalysis processes such as lactose hydrolysis and galacto-oligosaccharide synthesis in a high-salt industrial system.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a salt-activated β-galactosidase mutant D140G and its applications. Background Technology

[0002] β-galactosidase (EC 3.2.1.23) is a key member of the glycoside hydrolase family, possessing dual catalytic functions of β-1,4 glycosidic bond hydrolysis and transglycoside conversion. It exhibits significant application potential and practical value in the food, pharmaceutical, and biochemical industries. This enzyme can not only efficiently hydrolyze lactose, alleviating lactose intolerance, but also synthesize galacto-oligosaccharides (GOS) through transglycoside conversion. GOS are currently the only high-quality prebiotics that can be utilized by all beneficial gut bacteria in the human body, and are widely used in the development of functional foods and infant formula. Furthermore, in the dairy industry, this enzyme can be used for the resource utilization of lactose-free milk, yogurt, and whey waste; in food processing, it can be used for the catalytic conversion of fermented products; in the biopharmaceutical field, it can serve as a reporter gene tool enzyme and can also be used for the preparation of natural glycoside active substances; and in the biochemical field, it can participate in the synthesis of functional sugars and the catalytic conversion of biomass, covering multiple key industrial processes.

[0003] Currently, β-galactosidases still have significant shortcomings in practical applications, such as poor salt tolerance and difficulty in regulating enzyme activity. Salts are commonly used in industrial production systems to inhibit microbial contamination, but high concentrations of salt ions can disrupt the enzyme's spatial structure and interfere with the binding of the active site to the substrate, leading to a significant decrease in enzyme activity or even inactivation. Most β-galactosidases cannot maintain their catalytic activity in high-salt environments, limiting their direct application in such scenarios and requiring additional desalination processes, significantly increasing production costs and reducing efficiency. This makes it difficult to meet the demands of modern biomanufacturing for controllable, efficient, and stable catalytic processes.

[0004] β-galactosidases have a wide range of applications, but their activity is easily inhibited and difficult to regulate in high-salt systems. Therefore, developing novel β-galactosidases whose catalytic activity can be regulated by salt solutions is of significant research importance and application potential. These enzymes not only maintain catalytic activity in high-salt environments but also allow for dynamic regulation of enzyme activity by altering salt concentration. This enables catalytic reactions to be initiated, controlled, and runnable, truly transforming salt ions from inhibitory factors into regulatory tools. This characteristic can enhance the applicability and economy of enzymes in high-salt industrial systems, providing novel and efficient biocatalysts for food processing, biosynthesis, and the resource utilization of high-salt byproducts. β-galactosidases have a wide range of applications, but their activity is easily inhibited and difficult to regulate in high-salt systems. Therefore, developing enzymes whose catalytic activity can be efficiently regulated by salt solutions has significant research and application value.

[0005] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide a salt-activated β-galactosidase that can controllably regulate the efficiency of enzymatic reactions and is applicable to various technical applications where the rate of enzymatic reactions needs to be adjusted by the concentration of salt solution.

[0007] To achieve the above objectives, the present invention provides a salt-activated β-galactosidase mutant D140G, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] The present invention also provides a gene encoding the salt-activated β-galactosidase mutant D140G. d140g The nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

[0009] The present invention also provides a method comprising the said coding gene d140g The recombinant plasmid.

[0010] The present invention also provides a recombinant strain containing the recombinant plasmid.

[0011] Preferably, the recombinant strain is selected from Escherichia coli BL21(DE3).

[0012] The salt-activated β-galactosidase mutant D140G provided by the present invention can be used to catalyze the hydrolysis of lactose or p-nitrophenyl-β-D-galactopyranoside.

[0013] Preferably, the catalytic hydrolysis is carried out in the presence of a salt solution selected from one or more of NaCl, KCl, NH4Cl, NaBr, or KBr.

[0014] More preferably, the concentrations of NaCl and KCl in the salt solution are 0.05 M to 3.0 M, and the concentrations of NH4Cl, NaBr, and KBr are 0.5 M.

[0015] The present invention also provides a method for improving the activity of the β-galactosidase mutant D140G in lactose hydrolysis by adding salt to the reaction system to improve the activity of the mutant D140G in hydrolyzing lactose.

[0016] Preferably, the salt used in the method is 2 M NaCl or KCl.

[0017] The mutant D140G of this invention has the following advantages: The salt-activated β-galactosidase mutant D140G provided by this invention exhibits significant salt activation characteristics, with a specific activity of only 0.2 U / mg for pNPG under salt-free conditions, far lower than that of the wild-type enzyme 6Y2K. In 0.5 M solutions of NaCl, KCl, NH4Cl, NaBr, and KBr, the activity of the wild-type enzyme remains unchanged or slightly decreases, while the activity of D140G is significantly enhanced, increasing by 35.9–70.9 times in NaCl, KCl, and NH4Cl, and by 9.5–10.4 times in NaBr and KBr. In 0.05–3.0 M NaCl, the activity of D140G was significantly activated. At a NaCl concentration of 3.0 M, the pNPG hydrolytic activity reached 86.3 times that of the salt-free condition, while the wild-type enzyme showed no significant activation. In 0.05–3.0 M KCl, the D140G activity increased to a maximum of 103.3 times that of the salt-free condition, reaching a peak of 20.5 U / mg at 2.5 M. The relative activity of the wild-type enzyme decreased to 42.5% with increasing salt concentration. In experiments on the degradation of the natural substrate lactose, the activity of D140G was almost undetectable without salt. In 2.0 M NaCl and KCl, the specific activities reached 1.34 U / mg and 1.31 U / mg, respectively. Thin-layer chromatography confirmed that 2 M NaCl and KCl promoted lactose hydrolysis. Attached Figure Description

[0018] Figure 1 The results show the activity assays of the recombinant wild-type enzyme 6Y2K (a) and mutant D140G (b) in 0.5 M NaCl, KCl, NH4Cl, NaBr, and KBr.

[0019] Figure 2 The changes in the activity of recombinant wild-type enzyme 6Y2K (a) and mutant D140G (b) in NaCl solutions with final concentrations of 0.05–3 M are shown in this invention.

[0020] Figure 3 The changes in the activity of recombinant wild-type enzyme 6Y2K (a) and mutant D140G (b) in KCl solutions with final concentrations of 0.05–3 M are shown in this invention.

[0021] Figure 4 This invention relates to the difference in lactose hydrolysis activity of mutant D140G in NaCl (without salt) and KCl (final concentration of 2 M) in this invention.

[0022] Figure 5 TLC analysis of the lactose hydrolysis products of mutant D140G in this invention in NaCl and KCl at a final concentration of 2 M without salt. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Note: Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0025] Some of the experimental materials and reagents used in this invention: 1) Strains and vectors: The competent cells BL21(DE3) and expression vector pET-28a(+) used in this study were purchased from Beijing Qingke Biotechnology Co., Ltd.

[0026] 2) Enzymes and other biochemical reagents: p-nitrophenyl-β-D-galactopyranoside (pNPG) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; Nickel-NTA protein purification resin was purchased from QIAGEN; plasmid miniprep kit was purchased from Omega; QuickMutation... TM The site-directed mutagenesis kit was purchased from Beyotime Biotechnology Co., Ltd.; DpnI digestive enzyme was purchased from Beyotime Biotechnology Co., Ltd.; low molecular weight standard protein marker was purchased from Takara Biotech Ltd.; isopropyl-β-D-thiogalactoside (IPTG) was purchased from Solarbio Science & Technology Co., Ltd.; lactose was purchased from Tianjin Damao Chemical Reagent Factory; glucose assay kit was purchased from Nanjing Jiancheng Bioengineering Institute; thin-layer plates were purchased from Merck Group AG, Darmstadt, Germany; and all other reagents were of analytical grade.

[0027] 3) LB medium: Accurately weigh 10 g Trypone, 5 g Yeast extract, and 10 g NaCl, dissolve them in 1000 mL distilled water, and adjust the pH to natural (approximately 7.0). For solid media, add 2.0% (w / v) agar.

[0028] Example 1: Preparation of mutant D140G and construction and transformation of expression vector 1) Download the amino acid sequence of β-galactosidase 6Y2K (see SEQ ID NO.3, number: WP_100635792.1) and its corresponding nucleotide sequence from the GenBank database. 6y2k (See SEQ ID NO.4, number: NZ_NHTT01000002.1:199711-201681).

[0029] Amino acid sequence of 6Y2K (SEQ ID NO.3): MKLGVCYYPEHWPKSRWVEDAQHMRRIGIQYVRVGEFSWSTIEPTPGELHWEWLDESLDILHSQGLKVILGTPTATPPKWLVDRHPSMLAKDEAGRVRGFGSRRHYTFASLEYREECRRMVTMMAERYGHHPAVASWQTDNEYGCHDTVLSYAEADLAAFRLWLAEKYGTVEALNKAWGNVFWSMDYRSFDEIELPNLTVTEANPSHRLDFQRCCSDQVVAFNKLQVDILREHSAGRDLVHNYMGFFTAFDHHKVGQDLDVASWDSYPLGSLDKEPLYTEDEKHTYLRVGHPDAGAFHHDLYRGCGNGRLWIMEQQPGPVNWAPHNPTPADGAVRLWTWEAFSHGAELVSYFRWRQAPFGQEQMHAGLLRPDAQEAEAAKEATLVAQEVKVLAESIGLDADELMSLPSAGKVALMFDYDACWSLDIQPQSRAYRYFFWCYRMYEAMRELGLSVDIVPSNAPLDMYELLVLPAQAHITPELQNRLNSYQGVLLAGPRTGSKTETYQIPENLAPGPLASLLPLTVERVDALPEHTQPAVSGRWGAGKLKHWHEQIKTELPCLLKDDGGNPVLMGEGRHYYLGSCIDNTLLKASLAKLSEVAGLSTYYLPKGVRVRERGNVIFAFNYSSNTVVFEPQNAELVIGSMCLGAADVAIWKKQ。

[0030] 6y2k Nucleotide sequence (SEQ ID NO.4):

[0031] 2) Entrust Suzhou Hongxun Biotechnology Co., Ltd. to conduct 6y2k The sequences were optimized, modified, synthesized, and recombinant plasmids were constructed. First, the sequences were... 6y2k The sequence is optimized by adjusting the codon and GC content to obtain the optimized sequence. 6y2k-opt (See SEQ ID NO.5). In 6y2k-opt Two nucleotides (CC) are introduced before the start codon to form a sequence (CCATG) that has one less guanine nucleotide than the NcoI site (CCATGG) (to avoid frameshift mutations and ensure the normal progress of subsequent seamless cloning), and the stop codon is removed, thus obtaining... s6y2k-opt (See SEQ ID NO. 6), and synthesized it. To avoid introducing redundant nucleotide sequences into the recombinant wild-type enzyme gene sequence, a seamless cloning method was used to synthesize the wild-type enzyme. s6y2k-opt Ligated with the pET-28a(+) vector to construct recombinant plasmids s6y2k-opt -pET-28a(+) (The synthesis and expression of the 6×histidine His tag in β-galactosidase 6Y2K are terminated by a sequence on the vector).

[0032] Optimize sequence 6y2k-opt (SEQ ID NO.5):

[0033] s6y2k-opt (SEQ ID NO.6):

[0034] 3) Recombinant plasmid s6y2k-opt -pET-28a(+) was transformed into E. coli BL21(DE3) competent cells using the conventional heat shock transformation method, ultimately obtaining cells carrying pET-28a(+). s6y2k-opt Recombinant gene expression strain BL21(DE3) / s6y2k-opt This strain can express recombinant wild-type enzymes, the amino acid sequence of which is shown in SEQ ID NO.7.

[0035] The amino acid sequence of recombinant wild-type 6Y2K (SEQ ID NO.7): .

[0036] 4) The recombinant plasmid obtained above s6y2k-opt- pET-28a(+) strain BL21(DE3) / s6y2k- opt The plasmid was inoculated at a concentration of 0.1% into LB medium containing 50 μg / mL kanamycin and cultured overnight at 37 °C and 180 rpm. Plasmids were then extracted using a plasmid miniprep kit. s6y2k - opt -pET-28a(+).

[0037] 5) Using recombinant expression plasmids s6y2k-opt Using -pET-28a(+) as a template, mutant primers were designed using the Novizan online primer design website (https: / / crm.vazyme.com / cetool / singlepoint.html). The specific sequences are shown below. The designed primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0038] Mutation forward primer F (SEQ ID NO.8): gcagactggtaacgaatacggttgccacgaca; Mutant reverse primer R (SEQ ID NO.9): attcgttaccagtctgccaagaagcaacagcc.

[0039] Mutation was performed using the QuickMutation™ site-directed mutagenesis kit. The PCR reaction parameters were: 95°C pre-denaturation for 3 min; followed by 95°C denaturation for 30 sec, 55°C annealing for 30 sec, and 68°C extension for 8 min, repeated 20 times; 68°C extension completion for 15 min; and finally, cooling at 4°C for 30 min. PCR amplification yielded [the desired result]. d140g Recombinant plasmids of sequences d140g -pET-28a(+), this plasmid can express the mutant D140G in the host bacteria.

[0040] 6) Perform PCR product analysis Dpn I. Enzymatic digestion: Digest at 37 °C for 30 min to remove template DNA that has not undergone mutation.

[0041] 7) Transformation and Identification: The digestion products were transformed into E. coli BL21(DE3) competent cells using a heat shock transformation method to obtain cells carrying the digestion products. d140g Recombinant strain BL21(DE3) encoding the gene d140gSubsequently, sequencing was performed by Kunming Sangon Biotech Co., Ltd., and the sequencing results confirmed that the nucleotide sequence of mutant D140G is shown in SEQ ID NO.2, and its amino acid sequence is shown in SEQ ID NO.1. Compared with the amino acid sequence of the recombinant wild-type enzyme (SEQ ID NO.7), mutant D140G has a mutation at position 140 where aspartic acid is replaced by glycine, that is, the aspartic acid at position 140 of wild-type enzyme 6Y2K is replaced by glycine.

[0042] Note: D140 refers to aspartic acid at position 140 of recombinant wild-type enzyme 6Y2K (SEQ ID NO.7); D140G refers to the mutation of aspartic acid at position 140 of recombinant wild-type enzyme 6Y2K (SEQ ID NO.7) to glycine, and the amino acid sequence after mutation is shown in SEQ ID NO.1.

[0043] The amino acid sequence of mutant D140G (SEQ ID NO.1): MKLGVCYYPEHWPKSRWVEDAQHMRRIGIQYVRVGEFSWSTIEPTPGELHWEWLDESLDILHSQGLKVILGTPTATPPKWLVDRHPSMLAKDEAGRVRGFGSRRHYTFASLEYREECRRMVTMMAERYGHHPAVASWQTGNEYGCHDTVLSYAEADLAAFRLWLAEKYGTVEALNKAWGNVFWSMDYRSFDEIELPNLTVTEANPSHRLDFQRCCSDQVVAFNKLQVDILREHSAGRDLVHNYMGFFTAFDHHKVGQDLDVASWDSYPLGSLDKEPLYTEDEKHTYLRVGHPDAGAFHHDLYRGCGNGRLWIMEQQPGPVNWAPHNPTPADGAVRLWTWEAFSHGAELVSYFRWRQAPFGQEQMHAGLLRPDAQEAEAAKEATLVAQEVKVLAESIGLDADELMSLPSAGKVALMFDYDACWSLDIQPQSRAYRYFFWCYRMYEAMRELGLSVDIVPSNAPLDMYELLVLPAQAHITPELQNRLNSYQGVLLAGPRTGSKTETYQIPENLAPGPLASLLPLTVERVDALPEHTQPAVSGRWGAGKLKHWHEQIKTELPCLLKDDGGNPVLMGEGRHYYLGSCIDNTLLKASLAKLSEVAGLSTYYLPKGVRVRERGNVIFAFNYSSNTVVFEPQNAELVIGSMCLGAADVAIWKKQHHHHHH; The nucleotide sequence of mutant D140G (SEQ ID NO. 2)

[0044] Example 2 Preparation of recombinant wild-type β-galactosidase 6Y2K and mutant D140G 1) The two recombinant bacteria obtained in Example 1 (BL21(DE3) / s6y2k-opt BL21(DE3) / d140g The inoculum was 0.1% in LB (containing 50 μg / mL kanamycin) culture medium and activated by shaking at 37 ℃ and 180 rpm for 16 h.

[0045] 2) The activated bacterial culture was inoculated into fresh LB (containing 50 μg / mL kanamycin sulfate) culture medium at a 1% inoculation rate and cultured at 37 ℃ and 180 rpm for about 2-3 h until the OD600 reached 0.6-0.8. Then, IPTG was added to a final concentration of 0.7 mM for induction and cultured at 20 ℃ and 160 rpm for about 20 h to induce recombinant protein expression.

[0046] 3) After induction culture, the cells were collected by centrifugation at 4 ℃ and 6000 rpm for 8 min. The cells were then resuspended in an appropriate amount of 0.2 M McIlvaine buffer (pH=7.0) and sonicated under low-temperature water bath conditions. The homogenized cells were centrifuged at 12000 rpm for 30 min, and the supernatant was collected as the crude enzyme solution. The target protein was purified using a Nickel-NTAAgarose affinity chromatography column with an imidazole gradient elution from 0 to 500 mM.

[0047] 4) Add the protein sample obtained above to McIlvaine buffer (pH=7.0) at a volume ratio of 1:100 for dialysis. Cut the dialysis bag (mw: 14000) into appropriate lengths, boil in boiling water for 30 min, and then thoroughly wash with double-distilled water. Place the purified recombinant wild-type enzyme 6Y2K and mutant D140G obtained in step 3) into the dialysis bags respectively, leaving a certain length at both ends of the dialysis bag and sealing them with dialysis clips. Place the dialysis sample in the dialysis buffer and dialyze at 4 ℃. Change the dialysis buffer every 2 hours, for a total of 2 changes.

[0048] Example 3: Determination of the properties of recombinant wild-type β-galactosidase 6Y2K and mutant D140G The enzyme activity was determined using the pNP method, employing p-nitrophenyl-β-D-glucopyranoside (pNPG) as the substrate to measure the activity of the recombinant wild-type enzyme 6Y2K and mutant D140G prepared in Example 2. First, a pNPG solution with a final concentration of 2 mM was prepared and adjusted to the desired pH using buffer. The reaction system consisted of 50 μL of enzyme solution and 200 μL of 2 mM pNPG substrate. The substrate was preheated at the reaction temperature for 5 min, and then 50 μL of appropriately diluted enzyme solution was added. After reacting for 10 min, 750 μL of 1 M Na₂CO₃ was added to terminate the reaction. After the reaction mixture cooled to room temperature, the amount of released pNPs was measured at a wavelength of 405 nm. A control group was also included; the control group reaction system consisted of 50 μL of buffer and 200 μL of 2 mM pNPG substrate.

[0049] One enzyme activity unit (U) is defined as the amount of enzyme required to break down a substrate to produce 1 μmol of pNP per minute.

[0050] Methods for determining specific enzyme activity: The formulas for calculating enzyme activity (A) and specific enzyme activity (B) are as follows: A(U / mL)=c×N / (t×V); B (U / mg) = A / C; In the formula: c is the amount of p-nitrophenol after the enzyme reaction (μmol) calculated from the standard equation for p-nitrophenol; N is the enzyme dilution factor; t is the reaction time between the enzyme and the substrate (min); V is the volume of enzyme solution involved in the reaction (mL); and C is the protein mass concentration (mg / mL, determined using the BCA protein quantification kit).

[0051] 1) Activity determination of recombinant wild-type enzyme 6Y2K and mutant D140G in 0.5 M NaCl, KCl, NH4Cl, NaBr and KBr. 0.5 M salt solution was added to the enzymatic reaction system of the two enzymes. The enzymatic reaction was carried out with pNPG as substrate under the conditions of pH=6 and 37 ℃ for 10 min. The effects of different types of salts (NaCl, KCl, NH4Cl, NaBr and KBr) at the same concentration (0.5 M) on the enzyme activity of purified recombinant wild-type enzyme 6Y2K and mutant D140G were determined.

[0052] The activity assay results of recombinant wild-type enzyme 6Y2K and mutant D140G in NaCl are as follows: Figure 1 As shown, where, Figure 1 In the figure, 'a' represents the result of the assay for recombinant wild-type enzyme 6Y2K. Figure 1In the figure, b represents the measurement results of mutant D140G. The results show that the specific activity of mutant D140G under unsalted conditions (0.2 U / mg) is significantly lower than that of wild type (212.4 U / mg). However, in 0.5 M NaBr and KBr, the specific activity of D140G increased to 9.5 times and 10.4 times that under unsalted conditions, reaching 2.8 U / mg and 3.1 U / mg, respectively; in 0.5 M NaCl, KCl, and NH4Cl, its specific activity increased to 57 times, 71 times, and 36 times that under unsalted conditions, reaching 16.9 U / mg, 21.0 U / mg, and 10.7 U / mg, respectively. Compared with the mutant D140G, the recombinant wild-type enzyme 6Y2K showed no change or a slight decrease in activity under salt conditions. In 0.5 M NaCl, KCl, NH4Cl, NaBr and KBr, the relative enzyme activities were 91.9%, 85.7%, 60.9%, 101.7% and 86.8% of those without salt.

[0053] 2) Activity determination of recombinant wild-type enzyme 6Y2K and mutant D140G in NaCl 0.05–3.0 M NaCl was added to the enzymatic reaction system of the two enzymes. The enzymatic reaction was carried out with pNPG as substrate under the conditions of pH=6 and 37 ℃ for 10 min. The effect of different concentrations (0.05–3.0 M) of NaCl on the enzyme activity of purified recombinant wild-type enzyme 6Y2K and mutant D140G was determined.

[0054] The activity assay results of recombinant wild-type enzyme 6Y2K and mutant D140G in NaCl are as follows: Figure 2 As shown, where, Figure 2 In the figure, 'a' represents the result of the assay for recombinant wild-type enzyme 6Y2K. Figure 2 In the figure, b represents the measurement results of mutant D140G. The results show that the addition of 0.05~3.0 M NaCl to the reaction system significantly increased the enzyme activity of mutant D140G. In 0.05 M, 0.1 M and 0.3 M NaCl, its specific activity increased to 13.6 times, 14.0 times and 36.9 times that without salt, respectively, i.e. 2.9 U / mg, 3.0 U / mg and 7.9 U / mg. When the NaCl concentration in the system was 0.5 M~3.0 M, the relative enzyme activity increased to 4904.6%~8627.0%, and the specific activity reached 10.5~18.5 U / mg. In 3 M NaCl, the specific activity increased to 86.3 times that without salt, reaching a peak of 18.5 U / mg. In 0.05–3.0 M NaCl, the activity of wild-type enzyme 6Y2K did not change significantly, with the relative activity remaining between 75.4% and 117.2%.

[0055] 3) Activity determination of recombinant wild-type enzyme 6Y2K and mutant D140G in KCl 0.05–3.0 M KCl was added to the enzymatic reaction system of the two enzymes. The enzymatic reaction was carried out with pNPG as substrate under the conditions of pH=6 and 37 ℃ for 10 min. The effect of different concentrations (0.05–3.0 M) of KCl on the enzyme activity of purified recombinant wild-type enzyme 6Y2K and mutant D140G was determined.

[0056] 0.05–3.0 M KCl was added to the enzymatic reaction system of the two enzymes. The enzymatic reaction was carried out with pNPG as substrate under the conditions of pH=6 and 37 ℃ for 10 min. The effect of different concentrations (0.05–3.0 M) of KCl on the enzyme activity of purified recombinant wild-type enzyme 6Y2K and mutant D140G was determined.

[0057] The activity assay results of recombinant wild-type enzyme 6Y2K and mutant D140G in KCl are as follows: Figure 3 As shown, where, Figure 3 In the figure, 'a' represents the result of the assay for recombinant wild-type enzyme 6Y2K. Figure 3 In the figure, b represents the measurement results of mutant D140G. The results showed that the addition of 0.05~3.0 M NaCl to the reaction system significantly increased the enzyme activity of mutant D140G. In 0.05 M, 0.1 M and 0.3 M NaCl, its specific activity increased to 11.3 times, 15.2 times and 37.5 times that without salt, respectively, i.e. 2.2 U / mg, 3.0 U / mg and 7.5 U / mg. When the KCl concentration in the system was 0.5 M~3.0 M, the relative enzyme activity increased to 5053.6%~10313.3%, and the specific activity reached 10.0~20.5 U / mg. In 2.5 M KCl, the specific activity increased to 103.3 times that without salt, reaching a peak of 20.5 U / mg. In 0.05–3.0 M NaCl, the activity of wild-type enzyme 6Y2K decreased with increasing salt concentration, with a relative activity of 42.5%–91.6%. In 0.05 M KCl, the relative activity was 91.6%, and in 3 M KCl, the relative activity was 42.5%.

[0058] Example 4: Effects of NaCl and KCl on the activity of D140G mutant in hydrolyzing lactose and analysis of the products. 1) Determination of the lactose hydrolytic activity of mutant D140G under conditions without salt, NaCl, and KCl. Lactose hydrolysis activity was detected using the glucose oxidase-peroxidase (GOD-POD) method. In the reaction system, three groups were set up: no salt, 2 M NaCl treatment, and 2 M KCl treatment. The reaction was carried out at 37 ℃ for 90 min. After terminating the reaction by boiling in a water bath for 5 min, 2.5 μL of the reaction solution was collected for glucose quantification using a GOD-POD kit. The experiment was performed in triplicate, with an enzyme-free reaction system as a blank control to exclude non-specific degradation. Enzyme activity units were defined as the amount of enzyme required to catalyze the hydrolysis of lactose to produce 1 μmol of glucose per minute.

[0059] The results are as follows Figure 4 As shown, without salt, the activity of mutant D140G on lactose was almost undetectable, while in 2M NaCl and 2M KCl, the hydrolytic activity of mutant D140G on lactose was significantly increased compared with that without salt, with specific activities of 1.34 U / mg and 1.31 U / mg, respectively.

[0060] 2) Analysis of lactose hydrolysis products by mutant D140G in unsalted, 2 M NaCl, and 2 M KCl solutions The reaction products were analyzed by thin-layer chromatography. A Merck TLC silica gel plate (aluminum plate type) was used. The plate was dried in a 60 °C oven for 40 min before spotting. The spotting volume was 1 μL, and the developing solvent was n-butanol:ethanol:ultrapure water = 5:3:2 (v / v). Color development was performed using a diphenylamine system (1 g diphenylamine, 50 mL acetone, 1 mL aniline, 5 mL 85% phosphoric acid). After uniform soaking and drying, the plate was placed on a 100 °C hot plate for 10 min.

[0061] The results are as follows Figure 5 As shown, in the reaction systems of 2 M NaCl and 2 M KCl, the color intensity of lactose characteristic spots was significantly weakened, while the color intensity of glucose and galactose characteristic spots was significantly increased. In the reaction system without salt, the lactose characteristic spots maintained high color intensity, while no color was observed in the galactose and glucose regions. This indicates that 2 M NaCl and 2 M KCl significantly promoted the hydrolytic activity of D140G on lactose.

[0062] In summary, the β-galactosidase mutant D140G provided by this invention exhibits significant salt activation characteristics. The specific activity of this mutant in decomposing pNPG without salt is only 0.2 U / mg, far lower than that of the recombinant wild-type enzyme 6Y2K, but it can be significantly activated in various salt solutions. In 0.5 M NaCl, KCl, NH4Cl, NaBr, and KBr solutions, the enzyme activity of wild-type enzyme 6Y2K remains basically stable or slightly decreases, while the catalytic activity of mutant D140G is significantly enhanced. Its relative enzyme activity in NaCl, KCl, and NH4Cl reaches 35.9–70.9 times that without salt, and in NaBr and KBr it reaches 9.5–10.4 times. In NaCl solutions ranging from 0.05 to 3.0 M, the enzyme activity of mutant D140G was significantly increased, reaching a peak of 18.5 U / mg in 3.0 M NaCl, which is 86.3 times that under unsalted conditions. The relative enzyme activity of wild-type enzyme 6Y2K in NaCl solutions within this range remained only 75.4%–117.2%, showing no significant activation. In KCl systems ranging from 0.05 to 3.0 M, mutant D140G also exhibited significant salt activation, with its hydrolytic activity against pNPG increasing to a maximum of 103.3 times that under unsalted conditions, reaching a peak of 20.5 U / mg in 2.5 M KCl. In contrast, the activity of wild-type enzyme 6Y2K decreased continuously with increasing KCl concentration, reaching only 42.5% relative activity at 3.0 M. Experiments on the degradation of the natural substrate lactose showed that the hydrolytic activity of the mutant D140G was almost undetectable without salt. However, its hydrolytic activity towards lactose was significantly enhanced in 2.0 M NaCl and 2.0 M KCl, with specific activities reaching 1.34 U / mg and 1.31 U / mg, respectively. Thin-layer chromatography further confirmed that lactose could be hydrolyzed into glucose and galactose by D140G in the salted system, while no significant hydrolysis occurred without salt. These results indicate that the mutant D140G is a typical salt-activated β-galactosidase, exhibiting salt activation effects on both the artificial substrate pNPG and the natural substrate lactose, and possessing excellent catalytic potential under high-salt conditions.

[0063] Based on the significant salt activation characteristics exhibited by the D140G mutant, the rate of enzymatic reactions can be enhanced and controlled by adjusting the concentration of the salt solution in its reaction environment. This characteristic shows broad application prospects in the dairy industry, food processing, and biochemical engineering.

[0064] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A salt-activated β-galactosidase mutant D140G, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

2. The gene encoding the salt-activated β-galactosidase mutant D140G as described in claim 1. d140g Its characteristics are, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

2.

3. A recombinant plasmid, characterized in that, The recombinant plasmid contains the encoding gene as described in claim 2. d140g .

4. A recombinant bacterial strain, characterized in that, The recombinant strain comprises the recombinant plasmid as described in claim 3.

5. The recombinant strain according to claim 4, characterized in that, The recombinant strain was selected from Escherichia coli BL21(DE3).

6. The use of the salt-activated β-galactosidase mutant D140G as described in claim 1 in the catalytic hydrolysis of lactose or p-nitrophenyl-β-D-pyranogalactosidase.

7. The application according to claim 6, characterized in that, The application is carried out in the presence of a salt solution selected from one or more of NaCl, KCl, NH4Cl, NaBr, or KBr.

8. The application according to claim 7, characterized in that, The concentrations of NaCl and KCl in the salt solution are 0.05 M to 3.0 M, and the concentrations of NH4Cl, NaBr, and KBr are 0.5 M.

9. A method for improving the activity of the β-galactosidase mutant D140G as described in claim 1 during lactose hydrolysis, characterized in that, The activity of the mutant D140G in hydrolyzing lactose was enhanced by adding salt to the reaction system.

10. The method according to claim 9, characterized in that, The salt is 2 M NaCl or KCl.

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